Extractive purification of lipopeptide antibiotics

ABSTRACT

The present invention provides a rapid and inexpensive method for extractively isolating acidic lipopeptide antibiotics in high yield and purity.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation of allowed U.S. patentapplication Ser. No. 09/948,374, filed Sep. 6, 2001, which applicationclaims the benefit under 35 U.S.C. §119(e) of U.S. ProvisionalApplication Ser. No. 60/286,254, filed Apr. 24, 2001 and is acontinuation-in-part of U.S. patent application Ser. No. 09/760,328,filed Jan. 12, 2001, issued as U.S. Pat. No. 6,511,962 on Jan. 28, 2003,which claimed the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication Ser. No. 60/219,059, filed Jul. 17, 2000 and U.S.Provisional Application Ser. No. 60/220,950, filed Jul. 26, 2000. Theabove applications are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to methods of extractivelypurifying and/or isolating antibiotics and/or antimicrobial agents. Moreparticularly, the present invention relates to an inexpensive andefficient extractive method for purifying or isolating lipopeptideantibiotics.

[0004] 2. Description of the Related Art

[0005] An important class of antibiotics that inhibit gram-positivebacteria are the acidic lipopeptide antibiotics. Generally, acidiclipopeptide antibiotics consist of either a cyclic peptide core or acyclic depsipeptide core acylated with a lipophilic fragment and have anisoelectric point of less than about pH 7.0. The lipophilic fragment,typically an unsaturated fatty acid, may be of varying length.Frequently, the antibiotic activity of lipopeptide antibiotics isrelated to the length of the lipophilic fragment.

[0006] Examples of acidic lipopeptide antibiotics include, but are notlimited to, laspartomycin (Umezawa et al., U.S. Pat. No. 3,639,582;Naganawa et al., 1968, J. Antibiot, 21, 55; Naganawa et al., 1970, J.Antibiot., 23, 423), zaomycin (Kuroya, 1960, Antibiotics Ann., 194;Kuroya, JP 8150), crystallomycin (Gauze et al., 1957, Antibiotiki, 2,9), aspartocin (Shay et al., 1960, Antibiotics Annual, 194; Hausman etal., 1964, Antimicrob. Ag. Chemother., 352; Hausman et al., 1969, J.Antibiot, 22, 207; Martin et al., 1960, J. Am. Chem. Soc., 2079),amphomycin (Bodanszky et al., 1973, J. Am. Chem. Soc., 95, 2352),glumamycin (Fujino et al., 1965, Bull. Chem. Soc. Jap., 38, 515),brevistin (Shoji et al., 1976, J. Antibiotics, 29, 380), cerexin A(Shoji et al., 1976, J. Antibiotics, 29, 1268), cerexin B (Shoji et al.,1976, J. Antibiotics, 29, 1275) Antibiotic A-30912 (Hoehn et al., U.S.Pat. No. 5,039,789), Antibiotic A-1437 (Hammann et al., EP 0 629 636 B1;Lattrell et al., U.S. Pat. No. 5,629,288), Antibiotic A-54145 (Fukada etal., U.S. Pat. No. 5,039,789; Boeck et al., 1990, J. Antibiotics, 43,587), Antibiotic A-21978C (Debono et al., 1988, J. Antibiotics, 41,1093) and tsushimycin (Shoji et. al., 1968, J. Antibiot., 21, 439). Seealso Berdy, “CRC Handbook of Antibiotic Compounds,” Volume IV, Part I,pages 313-327, CRC Press, Boca Raton, FLA, (1980); Korzybinski et al.,“Antibiotics-Origin Nature and Properties,” Vol. 1, Pergamon Press, pp.397401 and 404-408, New York, N.Y. (1967).

[0007] The acidic lipopeptide antibiotics are typically active againstGram-positive microbes and constitute important therapeutics in thetreatment of infections caused by these bacteria. However, conventionalprocedures used to isolate and purify acidic lipopeptide antibioticsfrom fermentation broths involve a number of extraction andchromatography steps, which are time consuming, labor intensive andexpensive to carry out on a commercial scale. Thus, there is a need inthe art for improved methods of isolating and/or purifying acidiclipopeptide antibiotics.

BRIEF SUMMARY OF THE INVENTION

[0008] These and other needs are addressed by the present invention,which provides a rapid and inexpensive extractive method for purifyinglarge quantities of lipopeptide antibiotics in high yield. Quitesurprisingly, it has been discovered that acidic lipopeptide antibioticssuch as laspartomycin, amphomycin and aspartocin, which have cyclicpeptide nuclei and Antibiotic A-21978C, which has a cyclic depsipeptidenucleus, may under conditions of pH that are above the isoelectric pointof the lipopeptide antibiotic and in the presence of divalent metalcations such as Ca⁺², be directly extracted into water-immiscibleorganic solvents such as 1-butanol.

[0009] While not intending to be bound by any particular theory ofoperation, it is believed that acidic lipopeptide antibiotics formchelates with divalent metal cations such as, for example, Ca⁺² that arestable under basic conditions and that are soluble in water-immiscibleorganic solvents such as 1-butanol. Under acidic conditions, thechelates are disrupted and acidic lipopeptide antibiotics may beextracted into aqueous solution at basic or near neutral pH. Thus,according to one embodiment of the invention, the method comprisescontacting an aqueous composition comprising a lipopeptide antibioticand a divalent metal cation and having a pH above the isoelectric pointof the lipopeptide antibiotic with a water immiscible organic solvent,thereby extracting the lipopeptide antibiotic into organic solvent.Preferably, the pH of the aqueous composition is neutral or basic.

[0010] The lipopeptide antibiotic may then be extracted into aqueoussolution from organic solvent by acidifying the organic solvent at a pHbelow the isoelectric point of the lipopeptide antibiotic followed bycontacting the acidified organic solvent with an aqueous solution, whichis at neutral or basic pH. The lipopeptide antibiotic, which now behaveslike a conventional carboxylic acid, may be extracted back into organicsolvent by acidifying the aqueous solution and extracting the aqueoussolution with organic solvent. At this point, if necessary, thelipopeptide antibiotic may be further purified using extractive orchromatographic purification.

[0011] The extractive isolation methods of the current invention may beused to isolate and/or purity acidic lipopeptide antibiotics directlyfrom fermentation or culture broths, either before or after removal ofcells and/or cell debris and/or insoluble matter. Alternatively, theextractive isolation methods of the invention may be used in combinationwith conventional isolation and purification techniques. For example, anacidic lipopeptide antibiotic may be first precipitated fromfermentation or culture medium and the antibiotic isolated and/orpurified from the precipitate according to the extractive isolationmethods of the invention. The methods of the current invention may beused to advantageously isolate and/or purify synthetic acidiclipopeptide antibiotics and/or derivatives such as the syntheticlipopeptide derivatives described, for example, in Debono et al., 1988,J. Antibiotics, 41, 1093 and Lattrell et al., U.S. Pat. No. 5,629,288.

[0012] Thus, when used either alone or in combination with standardextraction and chromatographic techniques, the extractive methods of theinvention allow for the isolation of acidic lipopeptide antibiotics inhigh yield and high purity with fewer steps than are required byconventional methods.

DETAILED DESCRIPTION OF THE INVENTION

[0013] Reference will now be made in detail to preferred embodiments ofthe invention. While the invention will be described in conjunction withpreferred embodiments, it should be understood that it is not intendedto limit the invention to these preferred embodiments. To the contrary,it is intended to cover alternatives, modifications, and equivalents asmay be included within the spirit and scope of the invention as definedby the appended claims.

[0014] The extractive purification methods of the invention may be usedto rapidly and inexpensively isolate and/or purify virtually any acidiclipopeptide antibiotic. As used herein, “acidic lipopeptide antibiotic”refers to those antibiotics that have a cyclic peptidic nucleus with alipophilic fragment such as a fatty acid chain attached thereto and anisoelectric point of less than about pH 7.0. The cyclic peptidic nucleusmay be a cyclic peptide or a cyclic depsipeptide. The lipophilicfragment may be attached directly to the nucleus or through a linker,which is typically peptidic.

[0015] Acidic lipopeptide antibiotics may be natural products, syntheticor semisynthetic. Acidic lipopeptide antibiotics may also be derivativesof natural or synthetic acidic lipopeptide antibiotics, provided thatthe derivatives include carboxyl groups which permit extractiveisolation according to the principles of the current invention.

[0016] Exemplary acidic lipopeptide antibiotics which can beadvantageously isolated and/or purified according to the extractivemethods of the invention include, but are not limited to, laspartomycin(Umezawa et al., U.S. Pat. No. 3,639,582; Naganawa et al., 1968, J.Antibiot, 21, 55; Naganawa et al., 1970, J. Antibiot., 23, 423),zaomycin (Kuroya, 1960, Antibiotics Ann., 194; Kuroya, JP 8150),crystallomycin (Gauze et al., 1957, Antibiotiki, 2, 9), aspartocin (Shayet al., 1960, Antibiotics Annual, 194; Hausman et al., 1964, Antimicrob.Ag. Chemother., 352; Hausman et al., 1969, J. Antibiot., 22, 207; Martinet al., 1960, J. Am. Chem. Soc., 2079), amphomycin (Bodanszky et al.,1973, J. Am. Chem. Soc., 95, 2352), glumamycin (Fujino et al., 1965,Bull. Chem. Soc. Jap., 38, 515), brevistin (Shoji et al., 1976, J.Antibiotics, 29, 380), cerexin A (Shoji et al., 1976, J. Antibiotics,29, 1268), cerexin B (Shoji et al., 1976, J. Antibiotics, 29, 1275)Antibiotic A-30912 (Hoehn et al., U.S. Pat. No. 5,039,789), AntibioticA-1437 (Hammann et al., EP 0 629 636 B1; Lattrell et al., U.S. Pat. No.5,629,288), Antibiotic A-54145 (Fukada et al., U.S. Pat. No. 5,039,789;Boeck et al., 1990, J. Antibiotics, 43, 587), Antibiotic A-21978C(Debono et al., 1988, J. Antibiotics, 41, 1093) and tsushimycin (Shojiet al., 1968, J. Antibiot., 21, 439). See also Berdy, “CRC Handbook ofAntibiotic Compounds,” Volume IV, Part 1, pages 313-327, CRC Press, BocaRaton, FLA, (1980); Korzybinski et al., “Antibiotics-Origin Nature andProperties,” Vol. I, Pergamon Press, pp. 397401 and 404408, New York,N.Y. (1967).

[0017] Microorganisms that synthesize these various acidic lipopeptideantibiotics, as well as methods and conditions under which themicroorganism may be cultured to provide the various lipopeptideantibiotics are well known in the art (see e.g., Umezawa et al., U.S.Pat. No. 3,639,582; Debono et al., 1988, J. Antibiotics 41: 1093; Shayet al., 1960, Antibiotics Annual 194; Hamill et al., U.S. Pat. No.4,331,594; Hamill et al., U.S. Pat. No. 4,208,403; Hoehn et al., U.S.Pat. No. 4,024,245; Higgins et al., U.S. Pat. No. 4,024,246; Boeck etal., U.S. Pat. No. 4,288,549; Boeck et al., U.S. Pat. No. 4,994,270;Boeck, U.S. Pat. No. 4,977,083).

[0018] Those of skill in the art will appreciate that many acidiclipopeptide antibiotics are natural fermentation products comprisingmixtures of isomeric compounds. The various natural product isomersdiffer in one or more respects, typically in the lengths, branching,and/or degree of saturation of their respective fatty acid side chains.In other instance, such as the semisynthetic lipopeptide antibioticsdescribed in Debono et al., 1988, J. Antibiotics, 41, 1093 and Lattrellet al., U.S. Pat. No. 5,629,288 or those instances where natural productmixtures have been separated or where fermentation or culture conditionsare controlled such that a single type of molecule is produced, acidiclipopeptide antibiotic preparations will be “pure” with respect to theantibiotic molecule (i.e., it will not comprise a mixture of molecules).It should be understood that the extractive methods of the invention maybe used to isolate acidic lipopeptide antibiotics regardless of whetherthey constitute a mixture of molecules or a single type of molecule.However the methods of the invention do not separate different moleculesof a mixture of natural fermentation products from one another. Thus, ifthe producing microorganism synthesizes a mixture of acidic lipopeptideantibiotics, the extractive methods of the invention may be used toisolate the mixture from other contaminants and impurities.

[0019] Acidic lipopeptide antibiotics under normal conditions are notextracted into organic solvents, even polar organic solvents, fromneutral or basic aqueous solutions. Thus, when a neutral or basicaqueous solution of a acidic lipopeptide antibiotic is washed orcontacted with organic solvents, the lipopeptide antibiotic typicallyremains in the aqueous phase, as expected for a compound that containsmultiple carboxyl groups.

[0020] It has been discovered that under specified conditions, acidiclipopeptide antibiotics may be extracted into organic solvents fromneutral or basic aqueous solution, which enables easy separation fromacidic impurities by simple extraction. As is well known by those ofskill in the art, acidic lipopeptide antibiotics may be easily separatedfrom neutral and basic impurities by converting carboxyl groups of theantibiotic to carboxylate groups (i.e., by treating acidic lipopeptideantibiotics with base), extracting the carboxylate into aqueoussolution, converting the carboxylate groups back to carboxyl groups(i.e., by treating acidic lipopeptide antibiotics with acid), andextracting the carboxyl form into organic solvent. Thus, the method ofthe current invention, when used in conjunction with known methods ofextractively purifying lipopeptide antibiotics, enables isolation ofthese compounds freed from acidic, basic and neutral impurities in goodyield and high purity through simple extraction, which avoids the use ofexpensive and time consuming chromatography steps.

[0021] The conditions which permit an acidic lipopeptide antibiotic tobe partitioned or extracted into organic solvents from aqueous solutionunder neutral or basic conditions are related to the pH and the presenceof divalent metal cation such as Ca⁺² in the aqueous solution containingthe lipopeptide antibiotic. Generally, acidic lipopeptide antibioticsmay be extracted into organic solvents from aqueous solutions thatcontain divalent metal ion, which are maintained at a pH above theisoelectric point of the antibiotics.

[0022] While not wishing to be bound by any particular theory ofoperation, it is believed that adjusting the pH of the solution abovethe isoelectric point of the acidic lipopeptide antibiotic ionizes thecarboxyl groups. The carboxylate groups bind available divalent metal toform a stable divalent metal chelate of the antibiotic. The chelate,unlike the carboxylate anion of an acidic lipopeptide antibiotic, may beextracted into organic solvents from aqueous solution. Treating orwashing organic solvents that contain the chelate of an acidiclipopeptide antibiotic with acid disrupts the chelate, thus providingthe native acidic lipopeptide antibiotic.

[0023] Owing to this believed theory of operation, reference is madethrough the application to an “acidic lipopeptide antibiotic chelate.”However, it will be understood that this expression is being used merelyfor illustration and as a means for identifying a form of acidiclipopeptide antibiotic that can be extracted into organic solventsystems, and is not intended to be limiting in any way.

[0024] Acidic lipopeptide antibiotics may be isolated and/or purifiedaccording to the invention directly from fermentation and/or culturebroth, either with or without prior removal of cell debris.Alternatively, acidic lipopeptide antibiotics may first be isolated byconventional means, such as by acidic precipitation, and the precipitateresuspended and isolated and/or purified according to the extractivemethods of the invention. The methods of the invention may also be usedto isolate and/or purify synthetic acidic lipopeptide antibiotics and/orderivatives thereof.

[0025] As discussed above, a divalent metal cation may be chelated byionized carboxylate groups of the acidic lipopeptide antibiotic undercertain conditions. Thus, prior to extraction, the pH of the aqueoussolution comprising an acidic lipopeptide antibiotic should besufficiently basic to ionize the carboxyl groups of the lipopeptideantibiotic. Typically, the pH of the aqueous solution is adjusted to atleast a pH above the isoelectric point of the particular lipopeptideantibiotic being isolated. However, as the efficiency of the extractionis believed to depend on chelate formation, the pH of the aqueoussolution containing the lipopeptide antibiotic is ideally adjusted to avalue that is sufficiently basic to ionize all of the carboxyl groups ofthe lipopeptide antibiotic (i.e., at least about pH 5.0). Preferably,the pH of the aqueous solution is between about 7.0 and about 9.0, morepreferably, between about 8.0 and about 9.0 and most preferably, betweenabout 8.5 and about 9.0. Of course, if the acidic lipopeptide antibioticis extracted directly from fermentation or culture broth, the pH of thebroth may be sufficiently basic to render further adjustmentsunnecessary.

[0026] In order to form an acidic lipopeptide antibiotic chelate, theaqueous solution must include a divalent metal cation. Divalent metalcations that can form chelates with acidic lipopeptide antibiotics,which may be extracted into organic solvents according to the currentinvention include, but are not limited to, Ca⁺², Mg⁺², Zn⁺², Mn⁺², CU+²and Ni⁺². Preferred divalent metal cations include Ca⁺², Mg⁺² and Zn⁺².

[0027] The amount or concentration of the divalent metal cation is notcritical to success. However, since the method of the current inventionis believed to operate by chelating carboxylate groups of thelipopeptide antibiotic, the molar concentration of divalent metal cationrelative to the molar concentration of the acidic lipopeptide antibioticcarboxylate groups is at least about 0.5. Preferably, the molar ratio ofdivalent metal cation to carboxyl groups in the lipopeptide antibioticis between about 4:1 to about 10:1. If the number of carboxyl groups ina particular acidic lipopeptide antibiotic is unknown, a desireddivalent metal cation concentration and/or molar ratio may be readilydetermined empirically.

[0028] Divalent metal cation may be added to the aqueous solution by wayof salts, and may be added before or after culturing or fermenting theproducing strain, depending upon the requirement of the producingstrain. The identity of the counter anion(s) is not critical; however,if the salt is added prior to culturing or fermenting the producingstrain, a counter anion that negatively impacts the microorganismculture or fermentation broth should be avoided.

[0029] In many instances, the culture or fermentation broth and/orresuspended precipitate may contain a sufficient amount of divalentmetal cation such that addition of further cation may be unnecessary.Whether the addition of further divalent metal cation is necessary maybe determined by routine experimentation. Once the acidic lipopeptideantibiotic chelate has formed, it may be extracted into organic solventby contacting or washing the aqueous solution comprising the chelatewith organic solvent.

[0030] The organic solvent used to extract the acidic lipopeptideantibiotic chelate is not critical. However, it should satisfy twocriteria: first, it should dissolve appreciable quantities of the acidiclipopeptide antibiotic chelate (i.e., the acidic lipopeptide antibioticchelate should be more than sparingly soluble in the selected solventsystem) and second, it should be at least partially immiscible withaqueous solutions (i.e., the aqueous solution and the organic solventsystem should form two phases after mixing). Preferably, the organicsolvent is a polar solvent in which the acidic lipopeptide antibioticchelate is more than sparingly soluble, which is substantiallyimmiscible with water. The organic solvent may be a pure solvent or amixture of solvents. Suitable solvents and/or mixtures of solvents maybe identified by routine experimentation. A preferred organic solvent isn-butanol.

[0031] Calculating the volume of organic solvent needed to extract theacidic lipopeptide antibiotic chelate from aqueous solution is wellwithin the routine capabilities of skilled artisans. Typically, thevolume of the organic solvent ranges about ⅓ to about 3 times the volumeof the aqueous solution. Preferably, the volume of organic solvent usedto extract the acidic lipopeptide antibiotic chelate from aqueoussolution is about equal to the volume of the aqueous solution.

[0032] The aqueous solution may be contacted with as many portions oforganic solvent necessary to extract substantially all of the acidiclipopeptide antibiotic chelate into organic solvent. The number ofportions of organic solvent necessary to completely extract the acidiclipopeptide antibiotic chelate from aqueous solution (generally, atleast two portions of the organic solvent will be used to extract thelipopeptide antibiotic) may be readily determined by one of skill in theart.

[0033] Generally, contacting an aqueous solution containing an acidiclipopeptide antibiotic chelate with organic solvent in a separatoryfunnel is sufficient to extract the acidic lipopeptide antibioticchelate into organic solvent. However, in some situations, the organicsolvent and aqueous solution may be contacted by other methods wellknown to the skilled artisan such, as by magnetic stirring, mechanicalstirring, sonication, etc. Further, in some situations (i.e., inscale-up procedures) continuous liquid-liquid extraction may be used toextract the acidic lipopeptide antibiotic chelate into organic solvent.

[0034] The acidic lipopeptide antibiotic chelate may be disrupted bycontacting organic solvent containing the chelate with acid. Preferably,the organic solvent containing the acidic lipopeptide antibiotic metalchelate is contacted with an aqueous acid solution, most preferably,with aqueous mineral acid. Ideally, the pH of the aqueous acid solutionis sufficiently acidic to completely protonate the carboxylate groups ofthe acidic lipopeptide antibiotic chelate. Alternative methods ofprotonating carboxylate groups in organic solvent are known to theskilled artisan (e.g., saturating organic solvents with gaseous acidssuch as gaseous HCl or gaseous RBr, use of strong organic acids, etc.).Preferably, the pH of the aqueous acid solution is between about 3.0 andabout 1.0, more preferably, between about 2.5 and about 1.5 and mostpreferably, is about 2.0. While not wishing to be bound by anyparticular theory of operation, it is believed that acidifying theorganic phase protonates the carboxylate groups of the lipopeptideantibiotic chelate, thereby disrupting metal chelation by the carboxylicacid.

[0035] Once the organic phase containing the acidic lipopeptideantibiotic chelate has been acidified, the acidic lipopeptideantibiotic, which now behaves as a conventional carboxylic acid, may bepartitioned between organic solvent and aqueous acid and base solutionsby methods known to those of skill in the art. Thus, for example, theorganic solvent containing the free acid of the lipopeptide antibioticmay be contacted with aqueous base solution to provide an aqueousextract of a salt of the lipopeptide antibiotic Then, treatment of theaqueous solution of the salt of the lipopeptide antibiotic with aqueousacid solution allows extraction of the free acid of the lipopeptideantibiotic into organic solvent. The free acid of the lipopeptideantibiotic after treatment with neutral or basic aqueous solution, maybe again extracted into aqueous solution by conversion to the salt ofthe lipopeptide antibiotic. The acidic lipopeptide antibiotic may beisolated as either the salt or the free acid using methods known to theskilled artisan. The isolated acidic lipopeptide antibiotic may, ifdesired, be further purified by conventional methods well known to theskilled artisan such as silica gel chromatography, ion exchangechromatography, reverse phase chromatography, etc.

EXAMPLES

[0036] The invention having been described, the following examples arepresented to illustrate, rather than limit, the scope of the invention.The examples illustrate various embodiments and features of the presentinvention.

Example 1 Selection of Parent Culture for Laspartomycin

[0037] The parent culture used for biochemical synthesis oflaspartomycin is Streptomyces viridochromogenes ssp. komabensis,(ATCC-29814, BSP-M728) which was selected by the following procedure. Acell suspension of Streptomyces viridochromogenes ssp. komabensis,(ATCC-29814) was diluted so that plating on a nutrient medium gave wellseparated single colonies after incubation at about 28° C. A fewcolonies were isolated and tested by fermentation for improvement inlaspartomycin yield on the basis of morphological observations (colonysize, surface structure, edge profile, etc.) which are well within thecapabilities of those of skill in the art. The colony BSP-M728/1,provided higher and more reproducible yields and yielded superiorcorrelation with mycelial density in the fermentation mash. Thus, for atleast these reasons, Streptomyces viridochromogenes ssp. komabensis,(ATCC-29814, BSP-M728/1) was selected for biochemical synthesis oflaspartomycin.

Example 2 Medium Inoculation for Laspartomycin

[0038] Ideally, the biochemical synthesis of laspartomycin is performedby inoculating a medium composed of about 3.0% trypticase soy broth,about 1.0% corn dextrin and 0.1% CaCO₃ in tap water with spore andmycelial scrapings from a slant of Streptomyces viridochromogenes ssp.komabensis, (ATCC-29814, BSP-M728/1). Incubation of about 50 mL of theinoculated medium at 28° C. on a rotary shaker at about 200 revolutionsper minute (“RPM”) for about 48 hours provides a substantial and uniformvegetative growth. The growth may then be used to inoculate variousfermentation media (See, e.g., Example 3).

[0039] Preferably, the growth comprises a concentration range of betweenabout 2.0% to about 3.0% of the fermentation medium, when used toinoculate fermentation medium.

Example 3 Biochemical Synthesis of Laspartomycin

[0040] The inoculum produced in Example 2 may be used to seed a numberof fermentation media such as: (1) a medium containing about 2.0%dextrose, about 0.5% beef extract, about 0.5% peptone, about 0.5% NaCland about 0.35% CaCO₃ in water; (2) a medium containing about 0.5%dextrose, about 1.5% dextrin, about 1.0% molasses, about 1.0% peptoneand about 0.1% CaCO₃ dissolved in water; and (3) a medium containingabout 0.5% dextrose, about 1.5% glycerol, about 0.75% peptone, about0.2% NaCl and about 0.1% CaCO₃ in water. In typical shaker flaskfermentations, about 50 mL of the above media are seeded with theinoculum of Example 2 and are incubated at a temperature of about 28° C.on a rotary shaker at between about 160 and about 180 RPM for a periodof between about 4 and about 7 days.

Example 4 Biochemical Synthesis of Laspartomycin

[0041] Biochemical synthesis of laspartomycin may be performed in aculture medium containing about 0.5% dextrose, about 1.5% corn dextrin,about 0.75% Soytone, 0.3% NaCl, about 0.1% MgSO₄.7H₂O and about 0.1%CaCO₃ in water. The unadjusted pH of this medium is generally betweenabout 7.2 and about 7.3. The inoculated medium is incubated at atemperature of between about 24° C. to about 34° C. (preferably betweenabout 27° C. to about 29° C., most preferably about 28° C.) on a rotaryshaker at between about 140 and about 200 RPM (preferably between about160 and about 180 RPM) for a period of between about 4 and about 7 days(preferably, between about 5 and about 6 days) until significant amountsof laspartomycin are synthesized. Harvest pH readings of the medium arebetween about 8.0 and about 8.6. The yield for laspartomycin complex isabout 600 mg/liter of fermentation medium, while the yield of the C-15laspartomycin component is about 400 mg/liter of fermentation medium.The medium formulation and the quantitative ratio of its members has adirect effect on the ratio of the individual lipopeptide components oflaspartomycin.

Example 5 Biochemical Synthesis of Aspartocin

[0042] The biochemical synthesis of aspartocin is performed byinoculating a medium composed of about 1.0% dextrose, 0.5% molasses,1.0% Bacto Peptone, and 0.1% CaCO₃ in 100 mL of tap water, with sporeand mycelial scrapings from a slant of Streptomyces griseus ssp.spiralis (NRRL B-3290; BSP-M707). The inoculated medium is incubated ata temperature of about 28° C. on a rotary shaker at about 140 rotationsper minute (RPM) for about 48 hours providing a substantial and uniformvegetative growth. The latter may then be used to inoculate variousfermentation media as shown below; the concentration of vegetativegrowth when used to inoculate a fermentation medium ranges between 2.0and 3.0% of the fermentation medium. A number of fermentation media maybe successfully employed, such as: (1) a medium containing about 2.0%dextrose, 1.0% molasses, 1.0% Bacto-Peptone, and 0.1% CaCO₃ in 100 mL oftap water; and (2) a medium containing about 2.0% dextrose, 0.5%Bacto-Peptone, 1.0% Maltose, and 0.1% CaCO₃. In a typical shaker flaskfermentation, the above media are incubated at a temperature of about28° C. on a rotary shaker at about 140 RPM for a period of between about4 to about 7 days. Harvest pH readings are between 7.8 and 8.2.

Example 6 Biochemical Synthesis of Antibiotic A-21978

[0043] The biochemical synthesis of antibiotic complex A-21978 isperformed by inoculating a culture of Streptomyces roseosporus(NRRL-11379; BSP-M731) in a seed medium composed of about 3.0%trypticase soy broth, and 1.0% potato dextrin in 100 mL of tap waterfollowed by incubation at about 280 to 30° C. on a rotary shaker atabout 200 RPM for approximately 48 hours. The substantial vegetativegrowth provided by the above procedure may then be employed to inoculatea fermentation medium in a range of 2.0 to 3.0% of the fermentationmedium. A number of fermentation media may be successfully employed, butpreferably one containing about 0.75% dextrose, 3.0% potato dextrin,1.0% Soytone, 0.2% NaCl, 0.1% MgSO₄.7H₂O, and 0.25% molasses in 100 mLof tap water is used. In a typical shaker flask fermentation the aboveinoculated medium is incubated at a temperature of about 280 to 30° C.on a rotary shaker at about 200 RPM for a period of 4 to 7 days. HarvestpH readings range from about 6.0 to 6.5.

Example 7 Separation of Laspartomycin from Fermentation Broth withoutAddition of Divalent Metal

[0044] About 1.85 liters of fermentation broth produced as prepared inExample 4 (see e.g., Umezawa et al., U.S. Pat. No. 3,639,582) at pH ofabout 8.5 was mixed with an equal volume of 1-butanol and the phasesallowed to separate. The dark brown aqueous phase was discarded and theslightly colored 1-butanol phase containing laspartomycin was combinedwith an equal amount of distilled water, stirred and the pH of themixture was adjusted to about 2.0 with 1 N HCl. The 1-butanol phase waswashed with 1/4 its volume of water, mixed with an equal volume of waterand the pH of the mixture was adjusted to about 7.0. The phases wereseparated and the pH of the aqueous phase containing laspartomycin wasadjusted to about 2.0 and laspartomycin was extracted into 1-butanol andthen back into the aqueous phase at a pH of about 7.0. The aqueous phasecontained laspartomycin as the partial sodium salt. The solution wasevaporated under vacuum to remove residual 1-butanol and thenlyophilized to provide about 561 milligrams of the sodium salt oflaspartomycin as a white powder.

Example 8 Separation of Laspartomycin from Fermentation Broth withAddition of Diy Alent Metal

[0045] About 1.8 liters of fermentation broth as prepared in Example 4(see e.g., Umezawa et al., U.S. Pat. No. 3,639,582) was adjusted toabout pH 2.0 and allowed to stand at about 4° C. for three hours toprecipitate laspartomycin. The cells and precipitate were isolated bycentrifugation and suspended in about 500 mL of water. The pH of thesuspension was adjusted to about 7.0 with 1 N NaOH and the resultingmixture was stirred at room temperature for one hour. Calcium chloride(about 500 mg) was added to the suspension and the pH of the mixture wasadjusted to between about 8.6 and about 9.0 with 1.0 N NaOH.Laspartomycin was extracted from aqueous solution by two sequentialwashes with about 500 mL and then about 100 mL of 1-butanol. Thecombined butanol extracts were mixed with an equal volume of distilledwater, adjusted to about pH 2.0 with 1 N HCl and rinsed twice with about200 mL of distilled water maintained at about pH 2.0. The 1-butanolphase containing the antibiotic was separated, mixed with an equalvolume of distilled water and the mixture adjusted to about pH 7.0 with1 N NaOH to provide laspartomycin in the aqueous phase. The aqueousphase was separated and laspartomycin was then extracted into 1-butanolat about pH 3.0 and then into an aqueous phase at about pH 7.0. Theclear, almost colorless aqueous phase was evaporated under vacuum toremove residual 1-butanol and freeze-dried to obtain 668 mg of sodiumsalt of laspartomycin as a white powder. High resolution FAB-MS:calculated for C₅₇H₉₀N₁₂O₁₉+Na (M+Na)⁺: 1269.6343; found: 1269.6289.

Example 9 Separation of Laspartomycin from Fermentation Broth withAddition Of Diy Alent Metal

[0046] Calcium chloride (2.5 g) was added to 2.65 liters fermentationbroth as prepared in Example 4 (see e.g., Umezawa et al., U.S. Pat. No.3,639,582) at pH 8.7. The chelate of the laspartomycin complex wasextracted with 600 mL 1-butanol (phases were separated bycentrifugation). The cells and other material in an interface layer werere-extracted with another 100 mL of. 1-butanol. The 1-butanol phaseswere combined with 500 mL water and adjusted to pH 2.1 to removecalcium. The butanol phase, which contained laspartomycin, was washedwith 100 mL water (pH 2.0), separated from the aqueous layer, and thenmixed with 400 mL water adjusted to pH 7.5 to provide laspartomycin inthe aqueous phase. The aqueous phase was separated, adjusted to pH 2.3and mixed with 400 mL 1-butanol. The 1-butanol phase which, containedlaspartomycin, was washed with 100 mL water (pH 2.0) and then combinedwith 500 mL water and adjusted to pH 7.2. The aqueous phase, whichcontained laspartomycin as the partial sodium salt, was evaporated toremove residual butanol and freeze-dried to obtain 1.018 g of whitepowder, which appeared to be about 92% pure based on HPLC area % at 215nm. Approximately 79% of this complex was the major component,C₅₇H₉₀N₁₂O₁₉, at a retention time of 9.81 minutes. The minor componentshad retention times of 9.21 and 10.46 minutes. The HPLC system utilizeda Prodigy® 5μ ODS(2) column eluted with an eight minute gradient of 10%to 75% acetonitrile at pH 7.2 with 0.05 M phosphate buffer.

Example 10 Preparation of the Acid Form of Lasp Artomycin

[0047] About 100 mg of the sodium salt was prepared as described inExample 8. The sodium salt was then dissolved into about 10 mL of water,and the pH of the solution was adjusted to about 2.0 with 0.1 N HCl.Laspartomycin was then extracted into about 10 mL of 1-butanol. The1-butanol phase was washed with about 5 mL of water, mixed with about 20mL of water and evaporated under vacuum to obtain an aqueous solution oflaspartomycin in the acid form. This solution was freeze-dried to obtain77 mg of white powder. FAB-MS m/z:1248 (M+H)⁺, 1270 (M+Na)+ and 1286(M+K)+indicates a molecular formula of C₅₇H₉₀N₁₂O₁₉ for the C-15component of laspartomycin.

Example 11 Optimization of Calcium Concentration for Extraction ofAspartocin

[0048] The partial sodium salt of aspartocin 66 mg (˜0.05 mM), wasdissolved in 10 mL water to give a solution having pH 7.9. Calciumchloride, 5.5 mg (0.05 mM) dissolved in 0.11 mL water, was added alongwith 10 mL 1-butanol. The two phase system was shaken to equilibration.An aliquot of the 1-butanol phase, 0.25 mL, was removed for HPLCanalysis. Additional 5.5 mg of calcium chloride in 0.11 mL of water wasadded to the two phase system which was equilibrated after each additionand analyzed by HPLC. The HPLC system utilized a Prodigy 5μ ODS (2)column eluted with an eight minute gradient of 10% to 75% acetonitrileat pH 7.2 with 0.05 phosphate buffer. The maximum extraction of theaspartocin complex occurred when the approximate molar ratio of calciumchloride/complex reached 6. approximate molar ratio* HPLC area % at 215nm CaCl₂ (mg) CaCl₂ to complex for aspartocin complex 5.5 1 36% 11.0 271% 16.5 3 88% 22.0 4 92% 27.5 5 89% 33.0 6 100%  38.5 7 99%

Example 12 Extractive Purification of Aspartocin

[0049] Approximately 20 grams of a crude preparation of aspartocin (seee.g., Shay et al., 1960, Antibiotics Annual, 194), obtained by acidprecipitation of fermentation broth (see Examples 5 and 8) was mixedwith about 125 mL of water and insoluble impurities were separated bycentrifugation. About 300 mg of CaCl₂ was added to the brown coloredliquid and the resulting solution was adjusted to a pH of between about8.6 to about 9.0. Aspartocin was then extracted into about 100 mL of1-butanol. About 600 mg of CaCl₂ was added to the aqueous phase whichwas then extracted with another portion of 1-butanol. The combinedbutanol extracts were mixed with an equal amount of water, the pH of themixture adjusted to about 2.0 and the butanol phase washed with about160 mL of water adjusted to approximately pH 2.0. Aspartocin was thenextracted into water at about pH 7.0 and then back into butanol at a pHof between about 2.0 to about 3.0. The butanol phase was washed withabout 100 mL of water at approximately pH 2.0, then combined with anequal volume of water and adjusted to about pH 7.0. The aqueous phase isevaporated under vacuum to remove residual butanol. The very slightlycolored clear liquid was freeze-dried to obtain 803 mg of the sodiumsalt of aspartocin as a tan-white powder. Ions of the major component byFAB-MS m/z: 1340 (M+Na)⁺, 1384 (M+2Na—H)⁺, 1406 (M+3Na-2H)⁺, 1428(M+4Na-3H)⁺.

Example 13 Extractive Purification of Aspartocin

[0050] A dark colored crude preparation, 68.3 grams, containing 5-7% ofthe aspartocin complex in the acid form, was dissolved in 500 mLdistilled water and stirred as the pH was adjusted to pH 7.0. Someinsoluble material was separated by centrifugation and the decantedliquid was adjusted to pH 3.5. Aspartocin was extracted with twosequential 1-butanol extractions (500 mL, 300 mL) and 600 mL of waterwere added to the combined butanol extracts. The resulting two phasesystem was stirred and adjusted to pH 8.0 with 1 N NaOH to provide theaspartocin complex as the sodium salt in the aqueous phase. Calciumchloride (2.642 g) was added to the separated aqueous phase, andaspartocin extracted into 1-butanol as the chelate by two sequentialextractions (500 mL, 250 mL) of 1-butanol. The 1-butanol phases werecombined, mixed with 900 mL water, adjusted to pH 3.0, separated fromthe aqueous phase, and washed with 150 mL of water to remove calcium.The 1-butanol phase, which contained aspartocin was combined with 500 mLof water and adjusted to pH 7.0. To remove residual pigments, theaqueous phase containing the antibiotic was adjusted to pH 3.0 and mixedwith 500 mL of 1-butanol. The 1-butanol phase was separated, washed with150 mL water (pH 2-3), and combined with 500 mL water and the mixtureadjusted to pH 7.0. The aqueous phase, which contained aspartocin as apartial sodium salt was evaporated under vacuum to remove residual1-butanol and freeze-dried to obtain 3.6 g of a white powder. HPLCanalysis of the purified complex showed that the aspartocin complex wasapproximately 90% pure by 215 nm area % with peaks of the complexranging between 9.4 to 10.6 minutes retention time. The HPLC systemutilized a Prodigy® 5μ ODS(2) column eluted with an eight minutegradient of 10% to 75% acetonitrile at pH 7.2 with 0.05 M phosphatebuffer. The purified sample appeared to be approximately 98% pure byHPLC comparison to a reference sample of the aspartocin complex.

Example 14 Extractive Purification of Antibiotic A21978C

[0051] The cells from 1.9 L of fermentation broth were removed bycentrifugation. The decanted liquid (1600 mL) containing approximately204 mg of A21978C as determined by HPLC analysis was adjusted to pH 3.5with 1 N HCl and the antibiotic extracted into 600 mL butanol. Thebutanol was rinsed with 100 mL distilled water maintained at pH 3.5. The1-butanol phase containing the antibiotic was combined with 300 mLdistilled water and adjusted to pH 7.3 to provide Antibiotic A-21978C inthe aqueous phase. Calcium chloride (5 grams) was added to the aqueousphase and Antibiotic A-21978C chelate was extracted from the solution bytwo sequential extractions of about 250 mL each of 1-butanol. Thecombined 1-butanol extracts were mixed with an equal volume of distilledwater, adjusted to pH 3.5 with 1 N HCl and rinsed with 100 mL water, pH3.5 to remove calcium. The 1-butanol phase containing the antibiotic wasseparated from the aqueous phase and mixed with around 300 mL ofdistilled water. The pH was adjusted to 7.0 with 1 N NaOH to provide thepartial sodium salt of Antibiotic A-21978C in the aqueous phase. Theaqueous phase was evaporated under vacuum to remove residual 1-butanoland freeze-dried to obtain 176 mg of light tan colored powder. HPLCanalysis of the purified complex showed that the A21978C wasapproximately 83% pure by 215 nm area % with peaks of the complexranging between 7.9 to 9.9 minutes of retention time. The HPLC systemutilized a Prodigy® 5 m ODS(2) column eluted with an eight minutegradient of 10% to 75% acetonitrile at pH 7.2 with 0.05 M phosphatebuffer. The purified sample appeared to be approximately 90% pure by UVcomparison to reported values of E¹, cm of the A21978 components. FoundE^(1%) _(1 cm=)57 at 262 nm in EtOH, 41 at 280 nm, 36 at 290 nm and 26at 364 nm.

Example 15 Extractive Purification of Antibiotic A-21978C

[0052] Approximately 2.0 grams of a crude brown preparation ofAntibiotic A-21978C (see e.g., Debono et. al., 1988, J. Antibiotics, 41,1093) obtained by 1-butanol extraction of the fermentation broth (seeExample 6) was dissolved in about 150 mL water. About 1.0 grams ofcalcium chloride was added and the solution was adjusted toapproximately pH 8.7. The lipopeptide antibiotic was then extracted intoan equal volume of 1-butanol and the resulting aqueous phase wasre-extracted with about 50 mL of butanol. The two butanol extractionswere combined, mixed with an equal volume of water and adjusted withacid to about pH 2.0. The butanol phase was washed with about 150 mL ofwater at approximately pH 2.0. The lipopeptide antibiotic was thenextracted into water at about pH 7.0 and then back into butanol at a pHof about 2.0 to about pH 3.0. Antibiotic A-21978C was then extracted onefinal time into water at approximately pH 7.0 and evaporated undervacuum to remove residual butanol. The clear yellow solution wasfreeze-dried to obtain 160 mg of the free acid of Antibiotic A21978C asa light-yellow/tan powder. The original aqueous phase was extracted twomore times following the above procedure to provide an additional 260 mgof Antibiotic A-21978C as a light tan powder of similar quality.

[0053] While the invention has been described in some detail tofacilitate understanding, it will be apparent that certain changes andmodifications may be practiced within the scope of the appended claims.For example, different divalent metal ions, organic solvents, orlipopeptide antibiotics could be used in practicing the methods of thecurrent invention. Therefore, the above described embodiments should beconsidered illustrative and not restrictive and the instant invention isnot limited to the details given herein but may be modified within thescope of the appended claims.

[0054] All of the above U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, areincorporated herein by reference, in their entirety.

[0055] From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method for purifying a lipopeptide antibiotic, said methodcomprising the steps of: contacting an aqueous solution of thelipopeptide antibiotic and a divalent metal ion with an organic solvent,thereby extracting the lipopeptide antibiotic into the organic solvent;and contacting the organic solvent extract of the lipopeptide antibioticwith acid.
 2. The method of claim 1 in which the aqueous solution is afermentation broth or a culture.
 3. The method of claim 1 in which thelipopeptide antibiotic is a cyclic depsipeptide or a cyclic peptide. 4.The method of claim 1 in which the lipopeptide antibiotic is selectedfrom the group consisting of zaomycin, crystallomycin, amphomycin,glumamycin, daptomycin, antibiotic A-1437, antibiotic A-54145 andtsushimycin.
 5. The method of claim 1 in which the lipopeptideantibiotic is laspartomycin.
 6. The method of claim 1 in which thelipopeptide antibiotic is aspartocin.
 7. The method of claim 1 in whichthe lipopeptide antibiotic is antibiotic A-21978C or daptomycin.
 8. Themethod of claim 1 in which the pH of the aqueous solution of thelipopeptide antibiotic is adjusted to a basic pH.
 9. The method of claim8 in which the molar concentration of divalent cation relative tocarboxylate groups in the lipopeptide antibiotic is between about 4:1and about 10:1.
 10. The method of claim 2 in which the pH of thefermentation broth is adjusted to an acidic pH and cooled to about 4° C.11. The method of claim 10 in which the fermentation broth iscentrifuged and the centrifugate suspended in a second aqueous solution.12. The method of claim 10 in which the pH is about 2.0.
 13. The methodof claim 11 in which the pH of the second aqueous solution is adjustedto about pH 7.0.
 14. The method of claim 13 in which the molarconcentration of divalent cation relative to carboxylate groups in thelipopeptide antibiotic in the second aqueous solution is between about4:1 and about 10:1.
 15. The method of claim 14 in which the pH isadjusted to a basic pH.
 16. The method of any one of claims 8 or 15 inwhich the adjusted pH is in the range of about pH 8.0 to about pH 9.0.17. The method of any one of claims 9 or 15 in which the divalent cationis selected from the group consisting of Ca²⁺, Mg²⁺ and Zn²⁺.
 18. Themethod of claim 1 further comprising: extracting the lipopeptideantibiotic into a third aqueous solution; extracting the lipopeptideantibiotic into a second organic solvent; extracting the lipopeptideantibiotic into a fourth aqueous solution; and concentrating the aqueoussolution to provide a salt of the lipopeptide antibiotic.
 19. The methodof claim 18, wherein the organic extract of the lipopeptide antibioticis extracted into the third aqueous solution by washing with an aqueousbase solution.
 20. The method of claim 18, wherein the third aqueoussolution of the lipopeptide antibiotic is extracted into the secondorganic solvent by acidifying the third aqueous solution of thelipopeptide antibiotic and contacting with the second organic solvent.21. The method of claim 18, wherein the salt of lipopeptide antibioticis acidified to provide a free acid of lipopeptide antibiotic.
 22. Themethod of claim 21 in which the organic solvent and the second organicsolvent is 1-butanol.
 23. A method of isolating an acidic lipopeptideantibiotic, comprising the steps: contacting an aqueous solutioncomprising an acidic lipopeptide antibiotic with an organic solventunder conditions in which the antibiotic partitions into the organicsolvent; and recovering the antibiotic from the organic solvent.
 24. Amethod of isolating an acidic lipopeptide antibiotic, comprising thesteps of: (a) contacting an aqueous composition comprising thelipopeptide antibiotic and a divalent metal cation with an organicsolvent, wherein said aqueous composition has a pH above the isoelectricpoint of the lipopeptide antibiotic; (b) acidifying the organic phaseobtained from step (a); and (c) contacting the acidified organic phaseof step (b) with an aqueous solvent.